Our long term objective is to map the active sites of enzymes using activesite-directed enzyme-activated-inhibitors which can deliver highly active carbonium ions to the active sites. The full map for an enzyme will allow one to pinpoint the release point of the carbonium ion; systematic changes of variables will allow one to see how different inhibitors will have different release points (reflecting their different binding) and how those release points will change with pH, etc. With reference to chymotrypsin, the procedures used are: inhibition, reduction and alkylation, G-75 Sephadex separation of the peptides, sequencing of the peptide fragments formed as a result of O-alkylation of amide linkages, chemical modification of intact C-chain to increase solubility, tryptic and chymotryptic digestion, HPLC separation of peptides, full hydrolysis, amino acid analysis, synthesis of suspected N- benzyl amino acids, and chromatographic comparisons of the standards and unknowns to identify the latter. For the "mapping" of alpha-chymotrypsin, we have located the major site of alkylation on oxygen (carbonyl group of serine 214) and have made a practice run to determine the location of the stable labels (on N,S, and C); completion of the latter aspect - as a major thrust of this proposal - will complete the first case of mapping by our approach. A hypothesis had been developed to account for the activity of our D-family inhibitors in the inhibition of chymotrypsin. We plan to test that hypothesis in design and synthesis and testing of inhibitors for Trypsin. We will be using 13C NMR spectroscopy to guide us in the protein work, to identify certain benzyl substituted amino acids, and to follow chemical reactions carried out on the modified enzyme. Our method leads to the cleavage of peptide chains; nitrosolactam and nitrososultam inhibitors will be examined in an effort to control the site(s) of cleavage. Finally, attempts will be made to utilize fluorescent labels. The inhibitors we use are closely related to those with anti-cancer activity; further; anti-proteases are known to have potential uses in medicine.